The epidermis is one of the many stratified squamous epithelial tissues based on the keratinocyte cell type. Because of its accessibility for visual study and its propensity for undergoing neoplastic change, the epidermis has served as a model tissue for the study of epithelial tumorigenesis. A large body of literature now exists on the epidemiology, histology, and clinical course of epidermal cancer, and several preneoplastic lesions have been identified. Until recently, difficulty in culturing the keratinocyte has blocked detailed analysis at the cellular level in this otherwise well-defined system. Using a feeder layer of 3T3 mouse fibroblasts, normal human epidermal keratinocytes can be serially cultivated from low density inocula. The feeder layer apparently satisfies the physiological requirement of the keratinocyte for connective tissue support, and the several components of this requirement have now been identified in vitro. As is the case in vivo, cultured keratinocytes are subject to entering an irreversible program of terminal differentiation. The objective of the proposed research is to study in culture the keratinocytes from normal, benign, preneoplastic, and malignant lesions of the human epidermis. The cells will be evaluated with respect to the many tissue-specific in vitro attributes already established for normal keratinocytes. The aim is to define the cellular phenotypic markers associated with epidermal malignancy and preneoplasia so that the sequence of events in neoplastic development will be better understood. The properties of established lines derived in other laboratories from mouse epidermis, and the types of human carcinomas which have been cultivable, using traditional conditions (unfavorable for normal keratinocytes), suggest that some epidermal tumors lose their tissue-specific growth requirements. The hypothesis will be tested in this culture system that loss of keratinocyte-specific growth requirements is a necessary precondition for tumorigenicity in the epidermal keratinocyte.